Soil and sediment toxicity test methods currently in use (cf. Geisy, John P. and Robert A Hoke, Freshwater Sediment Toxicity Bioassessment: Rationale For Species Selection And Test Design, in J. Great Lakes Res. 15(4): 539-569) include:
a. the use of benthic organisms (which spend a significant portion of their lives in contact with sediments or soils) such as midges (mayfly larvae), clam larvae and earthworms, using parameters ranging from weight gain to percentage of test organisms surviving a timed exposure (days) to the solid particulate samples as the measure of toxic effect;
b. measurement of the toxic effects on algae;
c. determination of the effect on germination of seeds, and the growth rate of roots of sprouting seeds;
d. determination of the effect on fertilization of sea urchin eggs;
e. determination of lethality to higher organisms (e.g. mysid shrimp, dahpnia, fish) during a timed exposure (days), to either water or organic solvent (diluted with water) elutriates from the solid particulate samples;
f. changes in the light output of bioluminescent microorganisms when exposed to sample elutriates for relatively short times, such as 5 to 30 minutes (the Microtox.RTM. System, marketed by Microbics Corporation, Carlsbad, Calif.);
g. bioluminescent bacteria in direct contact with the solid particulate sample for about 20 minutes (the method of K. K. Tung, et al., described in U.S. patent application Ser. No. 07/682,923), which enhances response to sparsely soluble toxicants, a kit for the performance of which is currently being marketed by Microbics Corporation as the "Solid-Phase Test".
These methods, except the tests using benthic organisms, are all based on the well established art of testing water for acute toxicity. The most recent adaptation of a water acute toxicity test for the testing of solid particulate samples is the bioluminescent microorganism test utilizing direct contact between the solid particulate sample and the test microorganism, with filter separation of microorganisms from particulates before light readings are taken (Tung et al.). It is the most rapid and least expensive method available. In addition, this method typically results in toxic response data for a plurality of concentrations of the solid-phase sample, permitting the investigator to generate a dose-response curve, which is potentially a great advantage. A dose-response curve is a graph of the varying responses of a test organism to varying concentrations of the substance being tested. A reasonably smooth, monotonic curve is indicative of a definite functional relationship between the two variables. Traditionally, toxicologists have relied on the existence of such a dose-response curve not only for determining the toxicity quantitatively, but also as prima facie evidence that there is truly a causal relationship between the observed effect and the concentration of the substance tested. The quantitative result is usually reported as the LC50 (lethal concentration for 50% destruction) for aquatic organisms, and LD50 (lethal dose for 50% destruction) for mammalian toxicity results. For luminescent microorganisms, it has become conventional to report EC50, the effective concentration causing 50% light loss. More recently, however, the U.S. Environmental Protection Agency (EPA), Environment Canada and others have recommended making a distinction by using EC50 for "quantal" data (i.e. end-points resulting in a defined "effect", such as death, of 50% of the test organisms), and IC50 for the concentration causing a 50% "inhibition" of a measured functional parameter, a good example of which would be the reduction of light output from the luminescent bacterium, P. phosphoreum, in a Microtox.RTM. toxicity test. This Specification uses the IC50 convention, in anticipation of its general acceptance in the near future.
Data for several concentrations are not readily obtainable with tests employing benthic organisms. Due to the difficulty of performing tests with such organisms, they are most often exposed only to 100% of the test and reference samples, making the test semi-quantitative. Because of the speed and low cost of the luminescent microorganism test method of Tung et al., it is common practice to generate a dose-response curve for every test, providing additional assurance that there is a causative relationship. There are, however, several potential sources of error when bioluminescent microorganisms are mingled directly with solid particulate samples. This is true of both the method of K. K. Tung, et al., and the similar method described by H. Brouwer, et al., A Sediment-Contact Bioassay With Photobacterium Phosphoreum, in Environmental Toxicology and Chemistry, vol. 9, pp. 1353-1358, 1990.
One major source of concern is the difficulty of separating the particulates from the microorganisms before determining the toxic effect of the sample on the light output of the microorganisms, and another is the assurance that the relatively small sample being tested is truly representative of the total sample. Three major factors with regard to operational and theoretical difficulties are:
Factor 1; the possible optical interference of particulates, which cannot be totally separated from the microorganisms by filtration and/or by centrifugation;
Factor 2; the possible loss of microorganisms from the suspension due to adherence to the particulates at the time the separation is performed, whether by filtration, centrifugation or simple settling;
Factor 3; the demonstrable fact that the microorganisms solubilize, in effect, sparsely water-soluble toxicants in/on the solid particulate sample makes it very difficult to distinguish between a true toxic response and interferences from factors 1 and 2, above.
The uncertainties in data interpretation are compounded by the fact that "clean soils", soils which appear to be nontoxic when aqueous elutriates are tested, normally appear to be moderately toxic (e.g. 50% light loss for only one or two percent sample) when tested by the method of Tung et al. It is probable that some, if not all, of the light reduction is due to factors 1 and 2, above, but it is also quite possible that water-insoluble toxicants in and on the clean soil particles are bioavailable to the microorganism when in direct contact, and the clean soil is actually toxic to the bacteria. All three factors may be expected to depend on the physical properties of the particles, which means that soils from different locales may not have the same interference-to-real toxic response ratios. As a result, there has been some reluctance among users of this test to report the ICxx determined. (By convention, the ICxx is that concentration of sample which causes an xx % loss of light after a specified exposure time. IC50, a 50% light loss, is most often reported by users of the Microtox System.) A rigorous method for relating the results to those which the same sample would yield if it were free of interferences from factors 1 and 2, above, is lacking. This lack causes some users to treat the test results, which are otherwise inherently capable of providing quantitative answers, as if they are merely qualitative.
Using test and data handling approaches which are different from those of Tung et al., Brouwer et al. (p 1356, op.cit.) used only five toxicity ranks (severe, high, intermediate, low and very low) to characterize the toxicity of sediments they tested from Hamilton Harbor, Ontario, Canada. They tested only one fixed concentration and reported the toxic response of each sediment tested as the "% Photobacterium activity relative to control". The control sample was, in this case, an aliquot from a large (30 liter) sample collected from site 46, located four (4) or more km from the major sources of industrial pollution. The control sample (reference sample) and up to six other sediment samples were tested at the same fixed concentration, as a "batch", and the toxic response of each sample tested was assigned one of the qualitative toxicity ranks with respect to the toxicity of the control. The control sample was tested with every batch.
It is probable that all natural soils and sediments contain detectable quantities of sparsely soluble toxicants, such as metal salts and organics. It is reasonable, therefore, to be concerned with additional toxicity (e.g. added by man's activities) when surveying a site for toxicity. For example, the toxicity of soils/sediments relative to that of the pristine soil/sediment of the locale is the major interest when pollution assessment is the objective. The desired measurement in this case is the toxicity of each sample tested relative to that of the clean soil/sediment of the same locale. Until the present invention, there has been no method for determining the entire dose-response curve of a test sample relative to that of a reference sample, and the questions of how and where to obtain the correct clean soil/sediment reference sample at the beginning of a large survey project have not been answered in an economically feasible way. There has also been great difficulty in obtaining replicate samples for which there is confidence that they will be truly representative of the sample as a whole.
It is also desirable that the raw information for every sample tested be available, without any modification, to make the results relative to any other test. For example, a sample having a small unmodified IC50 (highly toxic) might be selected as the reference sample in the belief that it was improbable that it could have been contaminated by man. If a large number of samples of similar toxicity were to be compared to it by the method of Brouwer et al. the operator might conclude, erroneously, that they were all nontoxic. Upon discovery of the error, recovery could require retesting all samples using a new reference sample. Such a mistake could not occur if an IC50 were to be determined from raw data for every sample, assumed reference included, using the method of Tung et al. However, the IC50 values so determined would all be independent of each other, and prior to this invention there was no method for determining the relative toxicities from such independent test results.
As it has been practiced prior to this invention, the method of Tung et al. has used a very small (on the order of 0.4 grams) solid-phase sample. While the requirement for a very small sample has some advantages, it has been discovered that the sample size is a major factor contributing to the variance of IC50 in repeat tests of "the same sample". In effect, it has not been feasible to take a truly representative sample aliquot which is that small. The precision of the Tung et al. test is adequate for ranking of test samples, but it is marginal for purposes of determining the toxicity of samples relative to that of a reference sample of comparable toxic/interference responses, in accordance with the method of the present invention.
Similarly, when toxicity ranking alone is desired, it is not necessary to control the reaction temperature in practicing the invention of Tung et al. However, in practicing the method of the present invention, it is necessary to control the incubation temperature at the same value for all tests performed, in order to permit subsequent reduction of any and all data sets against any other as the reference sample. The preferred temperature is 15.degree. C., but any standardized temperature in the range of about 10.degree. to 30.degree. C. may be selected for use on a given toxicity survey program.
A similar need for relative toxicity determination exists in the field of acute and chronic water toxicity measurement by bioassay. There are many cases in which it is desirable to determine the toxicity of water before and after another source of toxicant is added, or before and after treatment intended to reduce the toxicity. Using the method of this invention, for example, the toxicity of the water in a stream could be determined relative to what it is/was on any given day for which data are/become available.
In summary, the current state of the art lacks a practical, economic means of rigorously determining the toxicity of a water or solid particulate sample relative to that of another sample. In particular, there has not been a method of generating a dose-response curve for one such sample relative to the other prior to this invention, nor has there been a method for confidently assuring that replicate samples were truly representative.